Glycans serve multiple functions which include assisting the folding of nascent proteins, determining the fate of cells and proteins in the circulation, and modulating developmental processes. The Golgi glycosyltransferases that are responsible for glycoconjugate synthesis are membrane-bound, but many of these enzymes are proteolytically cleaved in the Golgi, resulting in the release of the soluble catalytic domain. For some glycosyltransferases the soluble form is comparable to the membrane-bound counterpart in terms of its ability to form glycosylated products in vivo whereas for other glycosyltransferases the soluble form is markedly deficient as compared to its membrane-bound counterpart. This project will test the hypothesis that membrane-bound forms of Golgi glycosyltransferases preferentially glycosylate membrane proximal glycosylation sites on glycoconjugates whereas soluble forms of those same glycosyltransferases preferentially act on membrane distal sites. The substrate protein to be tested will be the mouse class I histocompatibility antigen, H-2Kb, whose three dimensional structure is known. Three mutant constructs of H-2Kb will be tested, each of which contains only one Asn-linked glycosylation site at either position 256 which is membrane-proximal or the membrane-distal positions 86 or 176. The test enzyme will be an isoform of alpha-2, 6 sialyltransferase (ST6Gal I) whose membrane-bound form has been shown not to be cleaved in the Golgi. In each experiment cells will be co-transfected with one of the H-2Kb mutant constructs and the cDNA encoding either the soluble form or the membrane-bound form of ST6Ga1 I. Extracts of transfected cells will be immunoprecipitated with antibodies specific for H-2Kb and then lectin blotted with the Sambucus nigra agglutinin (SNA), a lectin specific for a2, 6-linked sialic acid. Quantitation of the lectin blots, which will be normalized by Western blotting with anti-H-2Kb, will indicate the extent to which distance of glycan sites from the membrane effects the degree of glycosylation by membrane-bound versus soluble glycosyltransferases.